A particular receptor present on the surface of normal hepatocytes has strong binding with glycopeptides bearing terminal lactosyl or galactosyl group. By virtue of this property, 6-aminohexyl lactoside or 6-aminohexyl galactoside derivative conjugated complex is used as imaging agent for targeted receptors on hepatocytes (TWI 392511). The conventional method for preparing 6-aminohexyl lactoside comprises preparing acetobromo β-lactose in one step following the method described by Kartha (J Carbo Chem 9, 777-781, 1990), and preparing 6-trifluoroacetylaminohexyl β-lactoside following the method described by Weigel et al (Carbohydr Res 70, 83-91, 1979.). Briefly, β-lactose is treated with acetic anhydride and HBr/HOAc in a flask; and after acetobromo β-lactose is formed, the amount of the solvent is reduced, and diethyl ether is added for recrystallization, upon which acetobromo β-lactose as beautiful crystal is formed. Subsequently, 6-trifluoroacetylaminohexanol is reacted with acetobromo β-lactose with mercury cyanide as a catalyst, in toluene-nitromethane (1:1 by volume), to produce 6-trifluoroacetylaminohexyl β-lactoside.
The method for removing the acetyl group on acetyl β-lactose includes reacting the compound with a 10 mM solution of sodium methoxide in absolute ethanol for 2 hrs to remove the acetyl group, neutralizing the reaction product with acidic Dowex 50, and evaporating and drying the filtrate.
The method for removing the trifluoroacetyl group includes reacting overnight with 10% ethanol and 10% TEA (triethylamine) at room temperature. The mixture is suction dried, and the residue is then dried in a vacuum desiccators containing excessively amount of sodium hydroxide particles and concentrated sulfuric acid. To remove the counter ions of amino group, the residue is dissolved in 50% ethanol and passed through Dowex 1 (H+ type), until the liquid on the surface becomes basic. After filtering, the filtrate is suction dried.
The conventional glycosylation process of 6-aminohexanol includes acetylating lactose with acetic anhydride in the presence of pyridine.
Briefly, lactose (1.0 equiv, 20.0 g, 58.4 mmol) is added to a 500 mL reaction flask, a stirrer was placed, and then pyridine (180 mL) was added. The solution is stirred while in an ice bath, and acetic anhydride (12 equiv, 88 mL, 87.6 mmol) is added and stirred. After the reaction was complete as detected by TLC (upon which the solution appears clear and yellow), the solution is transferred to an extractor, and EtOAc (500 mL) was added. The impurities in the organic phase are removed by washing with 1 M HCl(aq) (400 mL×4), NaHCO3(aq) (200 mL×2), distilled water (200 mL), and saturated saline (200 mL).
It can be known from above that a 500 mL reaction flask and a 1 L extractor are needed for acetylating 20 g of starting lactose. However, as a result, deglycosylation generally occurs during deprotecting lactose by removing the acetyl group, such that the purification is difficult and the yield of the end product is low. Where the preparation process is scaled up, a quite large glass vessel and more solvent are needed, to alleviate the defect of low yield. When such large volumes of vessel and solvent are employed, not all of the experimental operators can run the reaction.
Taken up, in the conventional method above, the yield is about 10-15%, and deglycosylation generally occurs, which is not conducive to the subsequent massive production of 6-aminohexyl lactoside-NCS-benzyl-NODA-GA conjugate.